Some new nuclear magnetic resonance (NMR) approaches to elucidate chemical structures, which have not been determined by routine NMR methods, are presented. Selective detection of methine (CH), methylene (CH₂), or methyl (CH₃) signals in each subspectrum by editing NMR methods was utilized to reduce the complexity in crowded spectra. It also increased the peak separation and enhanced the sensitivity by limiting the measuring area of the 2D spectra. Several 2D methods to measure ^2,3J_CH values, which are useful for stereochemical assignment are then introduced. To determine the structure of a highly hydrogen-deficient molecule, efficient correlation methods for long-range ¹³C–¹³C coupling and ¹H-¹⁵N HMBC are also described.

View full abstract

Specific interaction between green fluorescent protein (GFP)-tagged human α- or γ-enolase^97-242 (α or γENO^97-242) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a “real-time FRET assay.” The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO^97-242 and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R_as) of ENO^97-242 mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.

View full abstract

Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer.

View full abstract

The insect antifeedant activities of pterocarpans and a sesquiterpene alcohol from the dichloromethane extract of Pterocarpus macrocarpus Kruz. (Leguminosae) were evaluated against the common cutworm, Spodoptera litura F. (Noctuidae), and the subterranean termite, Reticulitermes speratus (Kolbe)(Rhinotermitidae). Three pterocarpans, (−)-homopterocarpin (1), (−)-pterocarpin (2), and (−)-hydroxyhomopterocarpin (3) and the sesquiterpene alcohol, (+)-pterocarpol (5), were isolated from the dichloromethane extract of the heartwood of P. macrocarpus under guidance by a biological assay. Among these natural products, the most active insect antifeedant against both S. litura and R. speratus was 1. On the other hand, sesquiterpene alcohol 5 showed less insect antifeedant activity than the other pterocarpans against both insect species. While its methylated derivative, (−)-methoxyhomopterocarpin (4), showed high biological activity, 3 showed less insect antifeedant activity in this study. Interestingly, racemic 1 did not show insect antifeedant activity against S. litura. However, all of the test pterocarpans and isoflavones showed antifeedant activity against the test termites. Additionally, since these compounds were major constituents of P. macrocarpus, these antifeedant phenolics may act as chemical defense factors in this tree. In Thailand, lumber made from this tree is used to make furniture and in building construction due to its resistance to termite attack.

View full abstract

From the methanol extract of boron deficient Olea europaea leaves, two secoiridoid glycosides, not detected in leaf extracts of untreated plants, 6′-E-p-coumaroyl-secologanoside and 6′-O-[(2E)-2,6-dimethyl-8-hydroxy-2-octenoyloxy]-secologanoside, were isolated together with three known secoiridoid glycosides, oleuropein, oleoside dimethyl ester, and secologanoside. The structures of the isolated compounds were established by means of NMR and MS spectral analyses. The above novel secoiridoids were synthesized by the plant as a physiological response to nutrient stress.

View full abstract

The radical and superoxide scavenging activities of oxidized matairesinols were examined. It could be assumed that the free benzylic position was important for higher radical scavenging activity. The different level of activity was observed between 7′-oxomatairesinol (Mat 2) and 7-oxomatairesinol (Mat 3). The activity of 8-hydroxymatairesinol was lower than that of matairesinol (Mat 1). The superoxide scavenging activity of the oxidized matairesinols was also demonstrated for the first time. It is assumed that the pKa value of phenol in the oxidized matairesinols affected this activity.

View full abstract

To elucidate the structure-activity relationships of saponin on fruiting body induction in Pleurotus ostreatus, monodesmosidic saponins comprising a triterpenoid and glucose residues were synthesized. Betulin, a triterpenoid present in Birch bark, was used as the aglycon. The fruiting body induction activity increased with the number of glucose residues, from one to four. The most active betulinic saponin was betulin-3-yl β-D-(4-O-β-D-maltotriosyl)-glucoside.

View full abstract

A novel flavonol glycoside named camellianoside and three known flavonol glycosides were isolated from the leaves of Camellia japonica. The structure of camellianoside was established as quercetin-3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranoside by spectroscopic and chemical methods. The antioxidant activities of these glycosides evaluated by the diphenylpicrylhydrazyl (DPPH) radical scavenging reaction was higher than those of L-cysteine and L-ascorbic acid used as the reference antioxidants.

View full abstract

The transesterification of D-allose (the C-3 epimer of D-glucose) with vinyl octanoate using Candida antarctica lipase in tetrahydrofuran proceeded with high regioselectivity to produce 6-O-octanoyl-D-allose with nearly complete conversion. The growth-inhibiting activity of 6-O-octanoyl-D-allose on lettuce seedlings was about 6-fold greater than that of D-allose.

View full abstract

rcd1 is a mutant of Arabidopsis thaliana that is more resistant to methyl viologen, but more sensitive to ozone than the wild type. rcd1-2 is caused by a single nucleotide substitution that results in a premature stop codon at Trp-332. The rcd1-2 mRNA level does not change significantly with the mutation. Since overexpression of rcd1-1 cDNA has been shown to bring about an rcd1-like phenotype, we created and examined the overexpression lines of RCD1 by the use of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited a weak rcd1-like phenotype, although no resistance to methyl viologen was observed. Further, they fully complemented the aberrant rcd1-2 phenotype. Subcellular localization of RCD1 was examined by transiently expressing green fluorescent protein (GFP) fused with RCD1 in onion epidermal cells. GFP signals are observed as aggregated foci in the inner nuclear matrix-like region.

View full abstract

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

View full abstract

Octylphenol polyethoxylate (OPEO_n) biodegradation by Pseudomonas putida S-5 under aerobic conditions is initiated by the oxidation of its terminal alcohol group by alcohol dehydrogenase. A DNA fragment, containing an alcohol dehydrogenase gene (adh1), was isolated using a combination of degenerate PCR and inverse PCR. The predicted translation product of adh1 showed significant sequence similarity to bacterial alcohol dehydrogenases. Furthermore, a flavin-binding motif and signature patterns conserved in type III FAD-dependent alcohol oxidases were detected. Two open reading frames (ORFs) were found upstream of adh1, encoding a putative acyl-CoA synthetase and a putative esterase. Downstream of adh1 and located on the opposite strand was an ORF encoding a putative aldehyde dehydrogenase. Transcription analysis using RT-PCR showed that adh1 is cotranscribed with the putative acyl-CoA synthetase and esterase genes during growth on OPEO_n. ADH1 overproduced in Escherichia coli exhibited activity not only toward various alcohols, including OPEO_ns, but also toward primary aliphatic and aromatic aldehydes.

View full abstract

Soybeans are rich in pinitol (PI; 3-O-methyl-D-chiro-inositol), which improves health by treating conditions associated with insulin resistance, such as diabetes mellitus and obesity. Natto is a food made from soybeans fermented by strains of Bacillus subtilis natto. In the chromosome of natto strain OK2, there is a putative promoter region almost identical to the iol promoter for myo-inositol (MI) catabolic genes of B. subtilis 168. In the presence of MI, the putative iol promoter functioned to induce inositol dehydrogenase, the enzyme for the first-step reaction in the MI catabolic pathway. PI also induced inositol dehydrogenase and the promoter was indispensable for the utilization of PI as well as MI, suggesting that PI might be an alternative carbon source metabolized in a way involving the MI catabolic genes. Natto fermentation studies have revealed that the parental natto strain consumed PI while a mutant defective in the iol promoter did not do so at all. These results suggest that inactivating the MI catabolic genes might prevent PI consumption, retaining it in natto for enrichment of possible health-promoting properties.

View full abstract

In glycoside hydrolase family 66 (see http://afmb.cnrs-mrs.fr/CAZY/), cyclodextran glucanotransferase (CITase) is the only transglycosylation enzyme, all the other family 66 enzymes being dextranases. To analyze the catalytic amino acids of CITase, we modified CITase chemically from the T-3040 strain of Bacillus circulans with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). EDC inactivated the enzyme by following pseudo-first order kinetics. In addition, the substrates of an isomaltooligosaccharide and a cyclodextran inhibited EDC-induced enzyme inactivation, implicating the carboxyl groups of CITase as the catalytic amino acids of the enzyme. When two conserved aspartic acid residues, Asp145 and Asp270, were replaced with Asn in T-3040 mature CITase, CIT-D270N was completely inactive, and CIT-D145N had reduced activity. The V_max of CIT-D145N was 1% of that of wild-type CITase, whereas the K_m of CIT-D145N was about the same as that of the wild-type enzyme. These findings indicate that Asp145 and Asp270 play an important role in the enzymatic reaction of T-3040 CITase.

View full abstract

A novel glucanotransferase, involved in the synthesis of a cyclomaltopentaose cyclized by an α-1,6-linkage [ICG5; cyclo-{→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→}], from starch, was purified to homogeneity from the culture supernatant of Bacillus circulans AM7. The pI was estimated to be 7.5. The molecular mass of the enzyme was estimated to be 184 kDa by gel filtration and 106 kDa by SDS–PAGE. These results suggest that the enzyme forms a dimer structure. It was most active at pH 4.5 to 8.0 at 50 °C, and stable from pH 4.5 to 9.0 at up to 35 °C. The addition of 1 mM Ca²⁺ enhanced the thermal stability of the enzyme up to 40 °C. It acted on maltooligosaccharides that have degrees of polymerization of 3 or more, amylose, and soluble starch, to produce ICG5 by an intramolecular α-1,6-glycosyl transfer reaction. It also catalyzed the transfer of part of a linear oligosaccharide to another oligosaccharide by an intermolecular α-1,4-glycosyl transfer reaction. Thus the ICG5-forming enzyme was found to be a novel glucanotransferase. We propose isocyclomaltooligosaccharide glucanotransferase (IGTase) as the trivial name of this enzyme.

View full abstract

An extracellular phosphatase was purified to homogeneity from the entomopathogenic fungus Metarhizium anisopliae with a 41.0% yield. The molecular mass and isoelectric point of the purified enzyme were about 82.5 kDa and 9.5 respectively. The optimum pH and temperature were about 5.5 and 75 °C when using O-phospho-L-tyrosine as substrate. The protein displayed high stability in a pH range 3.0–9.5 at 30 °C and was remarkably thermostable at 70 °C. The purified enzyme showed high activity on O-phospho-L-tyrosine and protein tyrosine phosphatase substrate monophosphate (a specific substrate of protein tyrosine phosphatase). Although one peptide of the phosphatase shared identity with one alkaline phosphatase of Neurospora crassa, its substrate specificity and inhibitor sensitivity indicate that the enzyme is a protein tyrosine phosphatase.

View full abstract

We designed a new inverse PCR protocol combined with switching mechanism at 5′ end of RNA transcript (SMART) technology, and applied it to the cloning of teleost corticotropin-releasing hormone precursor cDNA. Due to the advantages of both techniques, this method can efficiently amplify the complete 5′- and 3′-ends of cDNA in a single reaction, and might prove to be an alternative to the conventional rapid amplification of cDNA ends (RACE) approaches.

View full abstract

Researchers working with fission yeast conduct protein extraction widely and frequently, but this includes the handling of glass beads, and hence is laborious and cumbersome, especially when dealing with a large number of samples. Here we describe a rapid and reliable method for preparing protein extract from fission yeast, one which is applicable to routine western blotting.

View full abstract

Four DNA fragments encoding a reverse transcriptase (RT)-like gene related to that of long terminal repeat (LTR) retrotransposons were isolated from the red alga Porphyra yezoensis by genomic PCR. Southern blot analysis suggested that one clone exists as a single copy per genome. Its full-length cDNA (PyRE2A) contained RT/RNase H-like sequences, which are most closely related to those of the Volvox LTR retrotransposon, although two stop codons were present within the RT region. We did not find any sequence related to LTR retrotransposons other than RT/RNase H in RyRE2A. These results indicate that PyRE2A is a single RT/RNase H-like gene and a defective progenitor of LTR retrotransposons.

View full abstract

Previously we reported the first example of peptide mimics of a small hydrophobic molecule, a phytohormone gibberellin. The second peptide mimic of gibberellin has been identified from random peptide libraries by its affinity to a type of catalyzing enzyme of gibberellins, which specifically recognizes bioactive gibberellins. These results suggest that even hydrophobic compounds can be mimicked by peptides.

View full abstract

A crude acerola polyphenol fraction (C-AP) was prepared by subjecting an acerola extract to a C18 cartridge column, and eluting the adsorbed fraction with ethanol containing 10% of acetic acid. C-AP appeared in a previous study to have an inhibitory effect on α-glucosidase and particularly on maltase activities. To elucidate the antihyperglycemic effect of C-AP further, we examined the regulation by C-AP of glucose uptake in Caco-2 cell; this resulted in the inhibition of glucose uptake. We next conducted single administration tests of glucose and maltose to ICR mice to investigate whether C-AP really controlled the intestinal glucose absorption in an animal body. The results showed that C-AP significantly suppressed the plasma glucose level after administering both glucose and maltose, suggesting that C-AP had a preventive effect on hyperglycemia in the postprandial state. The mechanism for this effect is considered to have been both suppression of the intestinal glucose transport and the inhibition of α-glucosidase. Despite such a preventive effect, the therapeutic effect of C-AP on hyperglycemia appeared to be low from the experiment with KKAy mice.

View full abstract

Abies koreana Wilson (A. koreana) is a shrub or broadly pyramidal evergreen tree endemic in the mountainous regions of South Korea. We obtained the essential oil (EO) from alpine needle leaves of A. koreana by the supercritical fluid extraction (SFE) method. EO was analyzed by gas chromatography–mass spectrometry (GC–MS), and 68 compounds were identified constituting 95.66% of the oil. The major components were elemol (11.17%), terpinen-4-ol (9.77%), sabinene (8.86%), 10(15)-cadien-4-ol (7.16%), α-terpineol (6.13%), α-pinene (6.07%) and γ-terpinene (4.71%). To investigate the memory-enhancing effects, we conducted a passive avoidance test using a scopolamine (1 mg/kg, ip)-induced amnesia mouse model. A peritoneal injection of EO from A. koreana (100 mg/kg) showed a memory enhancing effect of 72.7% compared with the control. These results suggest that EO of A. koreana may be a useful therapeutic agent against such amnesia-inducing diseases as Alzheimer and vascular dementia.

View full abstract

The characteristic aroma compounds of Citrus natsudaidai Hayata essential oil were evaluated by a combination of instrumental and sensory methods. Sixty compounds were identified and quantified, accounting for 94.08% of the total peel oil constituents. Limonene was the most abundant compound (80.68%), followed by γ-terpinene (5.30%), myrcene (2.25%) and α-pinene (1.30%). Nineteen compounds which could not be identified in the original oil were identified in the oxygenated fraction. Myrcene, linalool, α-pinene, β-pinene, limonene, nonanal, γ-terpinene, germacrene D, and perillyl alcohol were the active aroma components (FD-factor > 3⁶), whereas β-copaene, cis-sabinene hydrate and 1-octanol were suggested as characteristic aroma compounds, having a Natsudaidai-like aroma in the GC effluent. Three other compounds, heptyl acetate, (E)-limonene oxide and 2,3-butanediol, which each showed a high RFA value (>35) were considered to be important in the reconstruction of the original Natsudaidai oil from pure odor chemicals. The results indicate that 1-octanol was the aroma impact compound of C. natsudaidai Hayata peel oil.

View full abstract

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T_m, causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T_m^DSC, was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T_m of oTf by about 5–7 °C. However, neither sodium chloride nor sodium bicarbonate raised T_m by that much. Temperature T_m was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T_m^DSC, suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.
The Van’t Hoff plot from the T_m and ΔH_d values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T_m.

View full abstract

We found that soybean β-conglycinin peptone (BconP) suppresses food intake through cholecystokinin (CCK) release from enteroendocrine cells in association with binding of the peptone to rat small intestinal brush border membrane (BBM). The aim of the present study was to find new appetite suppressing peptides. Peptones from chicken, pork, beef, beef liver, and egg white were examined for activities to bind with rat BBM, CCK-release from enteroendocrine cell line STC-1, and induce satiety in rats. Chicken and pork peptone (ChickP and PorkP) bound to BBM with highest ability as evaluated with a surface plasmon biosensor. PorkP and ChickP released CCK in higher amounts than BconP from STC-1 cells dose-dependently, with highest stimulation by PorkP. An orogastric preload of PorkP, but not ChickP, suppressed food intake similarly to BconP, dose-dependently. These results suggest that PorkP interacts directly with the small intestinal CCK cells to release CCK, and that it suppresses appetite in rats.

View full abstract

The digestibility and gastrointestinal absorption of 14–16-kDa rice allergens (RAs) were investigated. RAs and bovine serum albumin (BSA) were first evaluated for their digestibility. BSA was digested completely by in vitro incubation with some proteases, but RAs remained almost intact. Administered orally (20 mg per mouse), intact RAs were clearly detected in the small intestine even 60 min after the administration, the amount of total RAs in the small intestine being estimated to be 0.59 mg. RAs were then biotinylated and infused into the duodenal lumen of anesthetized mice, and portal blood was collected. The RA concentrations in the portal plasma were respectively estimated to be 0.4–0.9 and 0.3–2.5 μg/ml for 0.4 and 4 mg doses. These results suggest that RAs are highly resistant to digestive enzymes and that about 1/100 of orally administered RAs remain intact in the small intestine, while at least 1/1,000–1/10,000 is absorbed and delivered into circulated blood.

View full abstract

Capsaicinol is an ingredient of hot red pepper. In this study, we developed a novel method for capsaicinol synthesis and examined capsaicinol’s physiological effects on capsaicin receptor (TRPV1)-related actions. Allylic oxidation of capsaicin by palladium acetate (Pd(OAc)₂) resulted in the formation of (±)-capsaicinol acetate at a 7.2% yield in a single step. The effectiveness of (±)-capsaicinol in TRPV1 activation (EC₅₀=1.1 μM) was found to be weaker than that of capsaicin (EC₅₀=0.017 μM), whereas the efficacy of (±)-capsaicinol reached 75% of that of capsaicin. Intravenous administration of (±)-capsaicinol in anesthetized rats dose-dependently enhanced adrenaline secretion from the adrenal gland. The response to a 5 mg/kg-dose of (±)-capsaicinol was comparable to that of a 0.05 mg/kg-dose of capsaicin. The relative pungency of capsaicinol to capsaicin was coincident with the relative effectiveness in inducing these TRPV1-related actions.

View full abstract

Phenolic acids such as p-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H⁺ cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase of p-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.

View full abstract

S-Allyl-L-cysteine (SAC), an active organosulfur compound derived from garlic, was found to reduce mortality with lesser incidence of stroke and also to lower the overall stroke-related behavioral score in stroke-prone spontaneously hypertensive (SHRSP) rats by dietary administration. Consequently, the anti-stroke effect of dietary SAC was demonstrated in SHRSP rats.

View full abstract

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N^τ-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.

View full abstract

The effect of dietary eritadenine on the plasma homocysteine concentration was investigated in methionine-induced hyperhomocysteinemic rats. The rats were fed on the control or eritadenine-supplemented (50 mg/kg) diet for 10 d. The animals were then injected with saline or methionine at a level of 100 or 300 mg/kg of body weight, and sacrificed 2 h or a more appropriate time after injection. The methionine injection increased the post-2 h concentration of plasma homocysteine in a dose-dependent manner in the control rats, this increase being significantly suppressed in the eritadenine-fed rats. This effect persisted up to 8 h after the methionine injection. The hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine were increased by eritadenine, whereas the hepatic homocysteine concentration was inversely decreased. The cystathionine β-synthase activity in the liver was increased by eritadenine. It is suggested from these results that eritadenine might suppress the methionine-induced increase in plasma homocysteine concentration by dual mechanisms: slowing the homocysteine production from S-adenosylhomocysteine and increasing the removal of homocysteine due to the enhanced activity of cystathionine β-synthase.

View full abstract

We have reported the antiallergic activities of the immunostimulatory oligodeoxynucleotide (ODN) BL07S, identified from genomic DNA of Bifidobacterium longum BB536 from in vitro and in vivo studies. The present study evaluated the efficiency of ODN BL07S in preventing allergic responses by oral administration. Oral administration of BL07S suppressed serum ovalbumin (OVA)-specific immunoglobulin (Ig) E levels and improved the OVA-specific IgG2a/IgG1 ratio. ODN BL07S increased Th1 cytokine and decreased Th2 cytokine production in splenocytes. These results suggest that immunostimulatory ODNs are potentially associated with the antiallergic effects of probiotics.

View full abstract

The nitrifying bacterium Nitrosomonas sp. strain ENI-11 has three copies of the gene encoding hydroxylamine oxidoreductase (hao₁, hao₂, and hao₃) on its genome. Broad-host-range reporter plasmids containing transcriptional fusion genes between hao copies and lacZ were constructed to analyze the expression of each hydroxylamine oxidoreductase gene (hao) copy individually and quantitatively. β-Galactosidase assays of ENI-11 harboring reporter plasmids revealed that all hao copies were transcribed in the wild-type strain. Promoter analysis of hao copies revealed that transcription of hao₃ was highest among the hao copies. Expression levels of hao₁ and hao₂ were 40% and 62% of that of hao₃ respectively. Transcription of hao₁ was negatively regulated, whereas a portion of hao₃ transcription was read through transcription from the rpsT promoter. When energy-depleted cells were incubated in the growth medium, only hao₃ expression increased. This result suggests that it is hao₃ that is responsible for recovery from energy-depleted conditions in Nitrosomonas sp. strain ENI-11.

View full abstract

We report here a development of the MultiSite Gateway^TM-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.

View full abstract

The ability to produce (R)- or (S)-β-phenylalanine from racemic β-phenylalanine through stereoselective degradation was screened for. Variovorax sp. JH2 and Arthrobacter sp. the faculty of Agriculture, Kyoto University (AKU) 638 were found to be potential catalysts for (R)- and (S)-β-phenylalanine production respectively. On 192 h cultivation of Variovorax sp. in medium containing 1.0% (w/v) racemic β-phenylalanine, 0.46% (w/v) (R)-β-phenylalanine with an enantiomeric purity of 99% e.e. was obtained. The initial step of the (S)-isomer degradation was stereoselective transamination. On 312 h cultivation of Arthrobacter sp. in medium containing 1.0% (w/v) racemic β-phenylalanine, 0.51% (w/v) (R)-β-phenylalanine with an enantiomeric purity of 90% e.e. was obtained. The initial step of the (R)-isomer degradation was supposed to be oxidative deamination. Resting cell reaction with vigorous shaking, with cells of Arthrobacter sp. as the catalyst, resulted in production of 0.49% (w/v) of (S)-β-phenylalanine with an enantiomeric purity of 99% e.e. from 1.0% (w/v) racemic β-phenylalanine in 45 h.

View full abstract

A growth-promoting substance (GPS) for methylotrophic yeasts was purified from yeast extract. The purified GPS showed growth promotion by reducing the lag time of the growth of methylotrophic Candida boidinii, while the growth rate at the exponential phase was the same as that of growth without GPS. GPS was finally identified to be L-proline by the criteria of instrumental analysis and chemical shift assignments.

View full abstract